Pneumocystis carinii infections remain common in HIV-infected patients despite the broad use of highly active antiretroviral therapies and prophylactic regimens. Studies of human P. carinii are focusing on two areas: diagnosis and evaluation for potential resistance to therapy. To try to develop highly sensitive, non-invasive diagnostic methods, we have been evaluating polymerase chain reaction (PCR) using primers based on the major surface glycoprotein (MSG) genes of human P. carinii. This is a family of genes that are closely related and that encode an important surface protein of P. carinii. PCR using primers based on this gene is potentially a highly sensitive method since this is a multicopy gene (estimated at greater than 100 copies/genome). We have been evaluating the diagnostic potential using a conserved region of the gene family. Our studies have shown that the sensitivity of MSG-based primers is greater than that of previously utilized primers. We are currently evaluating these primers prospectively in collaboration with the Microbiology Department and investigators at SFGH and the CDC. Because human P. carinii cannot be cultured, we cannot directly determine if resistance to commonly used therapeutic agents is developing. However, molecular techniques can be used to identify mutations that may confer resistance in genes that are targets of therapeutic agents. The most commonly used agent to treat P. carinii pneumonia is the combination of trimethoprim, which targets dihydrofolate reductase (DHFR), and sulfamethoxazole, which targets dihydropteroate synthase (DHPS). We have cloned the human P. carinii DHFR gene, and have examined (by PCR and sequencing) the P. carinii DHFR and DHPS genes of a variety of human isolates from patients with P. carinii pneumonia. DHPS mutations were found in about one-third of patients, while no mutations have been found to date in the DHFR gene. We have also expressed recombinant human P. carinii DHFR and characterized the kinetics of this enzyme. Over the past year we developed a rapid screening assay for agents which target DHFR by expressing the enzyme in a yeast system. We have also developed a rapid method for detection of DHPS mutations using SSCP (single strand conformational polymorphisms), and have examined a large number of samples for DHPS mutations, including organisms obtained from an Italian cohort. Over the past year we have developed and evaluated a new typing technique using tandem repeats that occur in an intron of the MSG gene. These studies should provide improved diagnostic methods for PCP and insights into the reasons for therapy or prophylaxis failures.